Three-dimensional scanning confocal laser microscope

ABSTRACT

A confocal microscope for generating an image of a sample includes a first scanning element for scanning a light beam along a first axis, and a second scanning element for scanning the light beam at a predetermined amplitude along a second axis perpendicular to the first axis. A third scanning element scans the light beam at a predetermined amplitude along a third axis perpendicular to an imaging plane defined by the first and second axes. The second and third scanning element are synchronized to scan at the same frequency. The second and third predetermined amplitudes are percentages of their maximum amplitudes. A selector determines the second and third predetermined amplitudes such that the sum of the percentages is equal to one-hundred percent.

Studies relating to this invention were funded, in part, by DOE grant #DE-FG02-91ER61229. The United States government has certain rights inthe invention.

This application is a division of application Ser. No. 08/683,607 filedJul. 15, 1996 (now U.S. Pat. No. 5,880,880, issued Mar. 9, 1999) whichis a continuation in part of application Ser. No. 08/372,479 filed Jan.13, 1995, now abandoned.

BACKGROUND

The invention relates to confocal microscopes.

Confocal microscopy involves focusing and scanning light at a sampleplane in order to form a high-resolution, highly magnified image.Conventional confocal microscopes image samples by generating a seriesof thin "optical sections" which are high-resolution images of thinplanes within the sample (see, e.g., U.S. Pat. Nos. 4,827,125;4,198,571, the contents of which are incorporated herein by reference).Confocal microscopy usually is done in vitro.

There are two conventional types of confocal scanning microscopes. Thetandem scanning confocal microscope ("TSCM") employs a rotating pinholedisk illuminated by a light source such as a mercury lamp. Duringoperation, the disk sweeps a focal spot through a sample, and isadditionally used to spatially filter back-scattered light. Thisinstrument has been used, for example, to image sections of skin. Asimilar device, the confocal laser scanning microscope ("CLSM"), uses alaser beam to image a sample, such as a biopsied tissue sample. In thisinstrument, the laser beam is focused to a nearly diffraction-limitedspot within a single plane of the sample. The spot is then scannedacross the plane, or alternatively, the sample is translated using amicrometer stage. In general, the CLSM has greater detection power,superior wavelength selectivity, and better illumination power than theTSCM.

SUMMARY

The invention features a confocal microscope including an image planerotation assembly. The image plane rotation assembly includes threescanning elements for scanning a light beam along three orthogonal axes.Two of the scanning elements are synchronized to scan at a selectedfrequency. A selector is used to set the desired amplitudes of thescanned light beams of the two synchronized scanning elements; thedesired amplitudes are a percentage of their maximum amplitudes and thesum of the percentages is equal to one-hundred percent.

Preferred embodiments include the selector being a controller with afrequency source for selectively varying the amplitudes of the twosynchronized scanning elements. The two synchronized scanning elementsare driven by a common frequency source.

The confocal microscope includes a pivoting articulated arm housing ascanning element at a pivot point of the arm, an imaging lens, and afocussing lens positioned between the second scanning element and theimaging lens. The focussing lens is configured to make an entranceaperture of the imaging lens an optical conjugate to the scanningelement in the arm so that spatial displacements of the field areminimized at the aperture. An aiming device aims the light beam as thelight beam exits the scanning element and is configured to position thescanning element such that the light beam remains substantially centeredon the entrance aperture of the imaging lens.

A light source emitting light at a wavelength between about 1550 and1800 nm produces the light beam.

According to another aspect of the invention, a coupler for coupling amicroscope objective lens to living tissue includes a template defininga tissue contacting surface for adhesive mounting to the tissue. Thecontacting surface defines a tissue imaging aperture. A housing definesa chamber for receiving the objective lens and includes a first mountfor rigidly coupling the objective lens to the housing and a secondmount for rigidly coupling the template to the housing.

According to another aspect of the invention, a method for generating animage of a sample includes illuminating the sample with a confocalmicroscope including an objective lens for focusing a light beam at thesample, and locating an index matching fluid having an index betweenabout 1.39 and 1.41 between the objective lens and the sample.

The inventions have many advantages. The operator can non-invasivelyview optical sections of living tissue which are oriented alonghorizontal, vertical, or variably-angled planes. Images can be takenfrom well below the surface of the tissue. The microscope's articulatedarm allows convenient positioning and contact with tissue. Thearticulated arm is configured to require fewer optical surfaces than inconventional systems, thereby minimizing light attenuation. The imaginglens contained by the arm can be placed in direct contact with thetissue, thereby minimizing relative motion between the lens and thetissue, and stabilizing the depth of the plane to be imaged.

Other features and advantages of the invention will be evident from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of a confocal microscopeaccording to the invention;

FIG. 1A is an enlarged view of a tissue engaging imaging lens of theconfocal microscope of FIG. 1;

FIG. 2 is a side-sectional view illustrating xy sections taken atintervals along the z axis;

FIG. 2A is a side-sectional view illustrating xz sections taken atintervals along the y axis;

FIG. 2B is a side-sectional view illustrating xw sections taken atintervals perpendicular to the w axis where w is an axis at an angle tothe y axis, in the yz plane;

FIG. 3 is a schematic of a circuit for adjusting the relative scanningamplitudes along the y and z axes;

FIG. 4 is a scan of a tilted plane;

FIG. 5 is a diagrammatic representation of an articulated arm of theconfocal microscope shown in two positions; and

FIG. 6 is a diagrammatic representation of an imaging lens coupler,according to the invention;

FIG. 7 is a diagrammatic representation of a mounting plate for use withthe imaging lens coupler of FIG. 6;

FIG. 8 is a plot of the absorption spectrum of water.

DETAILED DESCRIPTION

Referring to FIGS. 1 and 1A, a confocal microscope 10, according to theinvention, includes a laser 12 producing an imaging field 14 along anoptical path 15 (the z axis). Imaging field 14 is delivered to a tissue22, e.g, human skin, to be imaged via an articulated arm 20.

Imaging field 14 from laser 12 is propagated through a partiallytransmitting beam-splitting optic 34 onto a reflecting surface of a fastscan device 44, e.g., a rotating polygon mirror defining about 25reflecting facets driven at about 37,762 r.p.m. (this frequencycorresponds to 15.7 kHz, the standard video rate).

As fast scanner 44 rotates, imaging field 14 is scanned in anoscillatory fashion along an axis (the x axis) perpendicular to opticalpath 15 (the z axis). Imaging field 14 is then directed through atransfer lens 54 into articulated arm 20. Two representative fields 14',14" angularly displaced by fast scanner 44 are shown in the figures.

Articulated arm 20 houses a slow scanner 56, e.g., a galvanometricmirror oscillating at a frequency of 60 Hz. Oscillation of slow scanner56 scans the imaging field along a third axis (the y axis) perpendicularto the x and z axes. The scanned imaging field then passes through afocusing lens 66 onto a microscope objective lens 72. The depth of focuscan be adjusted with lens 72 or, alternatively, by a focusing lens 16(having, for example, a focal length of between 100 cm and 2 m) mountedon a scanning platform 18 which is translated along the beam path in anoscillatory manner in order to scan the depth of focus along opticalpathway 15 (the z axis). In FIG. 1A, the two representative fields 14',14" are shown focused within tissue 22 at discrete sites of constantdepth.

Imaging is preferably performed using light remitted (i.e., reflected oremitted light) from tissue 22. A small portion 14'" of the imaging field14 is reflected off of the tissue following irradiation. Remitted lightpasses through a pinhole 81 to an optical detector 80. The portion ofthe reflected field scanned along the x axis generates electricalsignals synchronized to the standard video frequency of 15.7 kHz, andthe portion of the reflected field scanned along the y axis and/or the zaxis generates electrical signals synchronized to a lower frequency ofabout 60 Hz, a rate corresponding to the frame-renewal frequency ofconventional U.S. video equipment.

Referring to FIGS. 2-2B, the scanning optics of the microscope can bemanipulated to allow imaging of a series of horizontal (xy) sections 100(FIG. 2), a series of vertical (xz) sections 110 (FIG. 2A), and a seriesof tilted (xw) sections 120 (FIG. 2B).

Different sections are imaged by changing the scanning magnitude (i.e.,the magnitude of the scanning angle or longitudinal displacement) of oneor more of the scanning optics 44, 56, 16. In the depth-dependenthorizontal sections 100, the x and y axes are scanned, while the z axisis translated incrementally. For imaging of vertical sections 110, the xand z axes are scanned, while the y axis is translated incrementally.

Tilted sections 120 are imaged by scanning all three axes, and adjustingthe relative scanning amplitudes of the y and z axes. Referring to FIG.3, the adjustments can be made using a balance control, such as a simplepotentiometer circuit, which allows the relative amplitudes of thewaveforms sent by a common waveform driver 58 to scanning platform 18and slow scanner 56 to be varied. The scan frequencies of scanningplatform 18 and slow scanner 56 are synchronized by driver 58 in afrequency range between about 1 and 100 Hz.

Circuit 105 allows scanning of the xy plane, the xz plane, or any planethat includes the x axis. The slow-scan drive voltage V is apportionedto drive amplifiers 202, 204 (z and y axes scanning) by a singlepotentiometer R through operational amplifiers 202a, 204a, respectively.For example, when the knob is all the way clockwise, then the fraction fis equal to zero, and the full voltage appears across drive amplifier202, resulting in an xz scan (the voltage across drive amplifier 204 isthen zero). When the knob is fully counter-clockwise, f is equal to one,and the full voltage appears across drive amplifier 204, resulting in anxy scan. In between, for example, setting f equal to 0.3, driveamplifier 204 is at 0.3 times (30% of) its maximum output and driveamplifier 202 is at 0.7 times (70% of) its maximum output, resulting inthe scan of a plane 201 at 23 degrees to the z axis (tan 23=0.3/0.7)(see FIG. 4).

Referring to FIG. 5, slow scanner 56 is positioned at pivot point 56a ofarm 20 located at the conjugate image plane corresponding to the objectplane of the fast scanner 44. This positioning minimizes movement ofimaging field 14 on the surface of a reflecting "elbow" joint 68. Duringoperation, slow scanner 56 undergoes two types of angular motion: rapid,oscillatory angular motion (at the frequency, e.g., of 60 Hz) scans theimaging field along the y axis for imaging purposes; while slow,non-oscillatory motion centers the imaging field on entrance aperture72' of objective lens 72 during movement of arm 20 (arrow 65). Slowscanner 56 is aimed on entrance aperture 72' by using a feedback sensor56b, e.g., an encoder, which senses the movement of arm 20 and then slowscanner 56 is moved by half the angle of rotation of arm 20 about pivotpoint 56a. The slow motion can be accomplished with a mechanical gearingapparatus, although electrically induced motion, such as galvanometricmotion, may also be used. The aperture of the objective lens 72 ispositioned at the conjugate image plane corresponding to the objectplane 64 of the mirror 56. The raster plane is shown at 70. The entranceaperture of the objective lens 72 is indicated by the lines 74.

Because the arm is mobile and can thus follow the movements of thesubject, small time-dependent fluctuations in the tissue, such as thosecorresponding to the subject's breathing or heart beat, can be correctedwhen the arm is secured to the subject.

Further stabilization of living skin can be achieved by more rigidmicroscope-to-skin coupling, e.g., by using an adhesive or suction torigidly fix the skin to the articulated arm of the confocal microscope.Referring to FIG. 6, microscope-to-skin coupler 300 includes a brassring 302 and template 304. Template 304 is glued onto the skin of thesubject with a hole 306 in template 304 aligned with the skin site to beimaged. A microscope objective housing 308 defines a chamber 310 whichreceives objective 72. Housing 308 includes observation windows 312 andmounting arms 314 defining bolt receiving holes 316.

In use, template 304 is glued onto the subject's skin and housing 308including objective 72 positioned within chamber 310 is placed withinring 302. Housing 308 and ring 302 are then attached using a screw 318.

Referring to FIG. 7, a mounting plate 320 includes a frame 322 defininga threaded hole 324 for engagement with a threaded portion 72a ofobjective 72. Arms 326 define slots 328 for attachment screws 330.Housing 308 is placed between arms 326 and screws 330 engage mountingholes 316 of arms 326. Slots 328 and slots 332 of arms 326,respectively, permit adjustment of the position of housing 308. Mountingplate 320 may be mounted to a three-dimensional stage (not shown) viaface plate 336.

Referring to FIG. 8, the optical wavelength of laser 12 is chosen tooptimize the image quality for a particular sample. Imaging of tissue 22is dependent upon the refractive index difference between neighboringstructures in the tissue and not absorption by stains or phosphorescentdies. This permits the wavelength of incident light to be selected formaximum penetration into the tissue. Wavelengths in the infrared aredesirable because the light is less scattered, but water in the tissueis highly absorbent. Wavelengths in the infrared between the major waterabsorption bands, e.g., at 1064 nm and 1650 nm, are therefore preferred.In general, preferred wavelengths for imaging are between 700 and 2000nm; shorter, visible wavelengths are strongly absorbed or opticallyscattered by most tissue, while selected bands and wavelengths deeper inthe infrared (i.e., 1940 nm and wavelengths greater than about 3microns) are more strongly absorbed by water present in most tissue.

Melanin, an epidermal absorber of light in the 600 nm to near-infraredregion of the spectrum, strongly backscatters light and thus can be usedwith the confocal microscope of the invention as a naturally-occurringcontrasting agent.

Refractive index matching of about 1.40 between objective lens 72 andtissue 22 enhances image quality. Forty percent sucrose in water as thematching fluid provides the desired index matching.

The confocal microscope may be used to generate, non-invasive,real-time, video-rate images of living tissue. Tissue can be imaged invivo and at depths in the range of about 0 to 3 mm in the skin; notissue biopsy or other invasive technique is required for obtaining aspecimen for microscopy.

Other embodiments are within the scope of the following claims.

What is claimed is:
 1. An articulated arm for use with a confocalmicroscope including a scanning element for scanning a light beam alongan axis, towards a sample said articulated arm comprising:a housingcontaining said scanning element said element being mounted at a pivotpoint of said housing, and an aiming device for aiming said light beamas said light beam exits said scanning element.
 2. The articulated armof claim 1 further comprising an imaging lens housed within said arm,said imaging lens being configured to receive the light beam from saidscanning element and focus the light beam to a focal plane in thesample, wherein said aiming device is configured to position saidscanning element such that said light beam remains substantiallycentered on an entrance aperture of said imaging lens.